The invention relates to a new expression vector for the production of a polypeptide in yeast, a yeast strain being transformed with such vector, and methods for the production of the vector, yeast strain and polypeptide.
Genetic engineering techniques for expression in yeasts commonly use shuttle vectors. The shuttle vectors have nucleotide sequences coding for a particular polypeptide combined with sequences necessary for expression in yeast, such as a yeast promoter. These shuttle vectors also have additional sequences that allow for expression in bacteria, such as Escherichia coli, or other microorganisms. Such additional non-yeast sequences are useful only for the construction of the vectors. However, they are superfluous for the expression in yeast. In fact they may hinder the efficient expression of the polypeptide in yeast or retard the replication of the organism because the superfluous nucleotides must also be doubled, which is an energy consuming process.
The yeast Saccharomyces cerevisiae is usually an excellent microorganism for the production of both homologous and heterologous proteins. This is because of its well characterized genetic system, rapid growth, and technical advantages of manipulation. Additionally, the development of DNA transformation systems for the introduction of cloned genes and their inexpensive and safe overproduction in simple fermentation conditions, has made this organism particularly useful for large-scale industrial practice.
A number of yeast polypeptides are known in the art. Of particular interest are the superoxide dismutases. The yeast Saccharomyces cerevisiae contains two species of superoxide dismutases (EC 1.15.11), the copper/zinc-(Cu/Zn SOD) and the manganese-(Mn SOD) containing forms. The Cu/Zn SOD is localized in the cytoplasm while the manganese enzyme is restricted to the mitochondrial matrix. This enzyme is assumed to provide in vivo protection against toxic free radicals generated within cells as intermediates of normal metabolism (Bilinski, T. et al. Biochem. Biophys. Res. Commun. 130: 533-539 (1985), Van Loon A.P.G.M. et al. Proc. Natl. Acad. Sci. USA 83: 3820-3824 (1986), Lee F. J. et al. J Free Rad. Biol. Med. 1:3 19-325 (1985), Galiazzo F. et al. Biochim. Biophys. Acta 965: 46-51 (1988)). Consequently, it is expected to be useful for preventing or treating potential damage in human, particularly damage from cell aging and senescence (Rosen D. R. et al. Nature 362, 59-62 (1993), McCord J. M. and Fridovich I. J Biochem 244: 6049-6055 (1969),. McCord J. M. et al. Proc. Natl. Acad. Sci. USA 68: 1024-1027 (1971), McCord J. M. N. Engl. J Med. 312: 159-163 (1985)).
The Cu/Zn SOD gene from Saccharomyces cerevisiae was cloned, sequenced (Bermingham-McDonogh 0., et al. Proc. Nat. Acad. Sci. USA 85: 4789-4793 (1988)), and the structure and mechanism of action of the enzyme is well characterized (Djinovic K. et al. J. Mol. Biol. 225: 791-809 (1992), O""Neill P. et al. Biochem. J 251: 41-46 (1988)). The Cu/Zn SOD is an abundant metalloenzyme present in the cytoplasm of most aerobic and many anaerobic organisms, whose activity catalyzes the dismutation of the superoxide anion to dioxygen and hydrogen peroxide.
It is an object of the present invention to improve on the yields of polypeptides in the fermentation processes of yeasts transformed with expression vectors coding for such polypeptides. It is a further object to provide new vectors which are able to express desired polypeptides in yeast in larger amounts as compared to previous processes. A further object is to provide new yeast strains transformed with such vectors that are superior compared to the wild-type strain or those which are transformed with shuttle vectors.
The present invention provides for expression vectors and a yeast strains. In particular a Saccharomyces cerevisiae strain transformed with a vector, which produces higher levels of yeast or non-yeast polypeptides compared to the wild-type strain or those transformed with a shuttle vector. Methods for the preparation of such expression vector, yeast strains and endogenous yeast polypeptides are set forth.
Numerous aspects and advantages of the invention will be apparent to those skilled in the art upon consideration of the detailed description and the drawings of the invention which provides illustrations of the practice of the invention in its embodiments.